FEBRUARY 2000 VOLUME 48, NUMBER 2
© Copyright 2000 by the American Chemical Society
Biotransformation of β-Myrcene by the Larvae of Common Cutworm (Spodoptera litura) Mitsuo Miyazawa* and Takeshi Murata Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
β-Myrcene was mixed in an artificial diet at a concentration of 1 mg/g of diet, and the diet was fed to the last instar larvae of common cutworm (Spodoptera litura). Metabolites were recovered from frass and analyzed spectroscopically. β-Myrcene was transformed mainly to myrcene-3(10)-glycol and myrcene-1,2-glycol. Each pair of double bonds of β-myrcene was converted to the corresponding diol by oxidation, respectively. The 3,10- and 1,2-double bonds of β-myrcene were respectively oxidized. Keywords: Common cutworm; Spodoptera litura; biotransformation; β-myrcene; myrcene-3(10)-glycol; myrcene-1,2-glycol INTRODUCTION
Terpenoids are known as not only raw materials for flavor and fragrance but also biologically active substances. A great majority of biologically active terpenoids are produced as plant secondary metabolites, and these terpenoids have been shown to have biological activity against plants, microorganisms, and insects. Various attempts have been made to search for new biologically active terpenoids. Biotransformation is one of way to produce biologically active terpenoids. Previously we reported biotransformation of R-terpinene and (+)- and (-)-limonene by the larvae of common cutworm (Spodoptera litura) (Miyazawa et al., 1996, 1998). These studies revealed that the C-7 position (allylic methyl group) of R-terpinene was preferentially oxidized. The results indicated that the intestinal bacteria probably participated in the metabolism of R-terpinene. Then (+)- and (-)-limonene were oxidized at the 8,9-double bond and the C-7 position (allylic methyl group). * Author to whom correspondence should be addressed (telephone +81-6-6721-2332; fax +81-6-6727-4301; e-mail
[email protected]).
There have been no studies on the biotransformation of β-myrcene (1), an acyclic monoterpene, by the larvae of S. litura. In the present paper, the biotransformation of β-myrcene by the larvae of S. litura was investigated for the purpose of estimating possible metabolic pathways in insects. Compound 1 is a typical acyclic monoterpene. It is found in bay oil, verbena oil, and hop oil, has a balsam-like sweet odor, and is widely used in beverages. This paper deals with the metabolism from frass and the metabolic pathways. MATERIALS AND METHODS Chemicals. The β-myrcene (1) was purchased from Taiyo Perfume Co., Ltd. (Osaka, Japan). Gas Chromatography (GC). A Hewlett-Packard 5890A gas chromatograph equipped with a flame ionization detector, an HP-5MS capillary column (30 m length, 0.25 mm i.d.), and a split injection of 20:1 were used. Helium at a flow rate of 1 mL/min was used as a carrier gas. The oven temperature was programmed from 80 to 240 °C at 4 °C/min. The injector and detector temperatures were 250 °C. The peak area was integrated with a Hewlett-Packard HP3396 SERIES2 integrator. Gas Chromatography/Mass Spectrometry (GC-MS). A Hewlett-Packard 5890A gas chromatograph equipped with a
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124 J. Agric. Food Chem., Vol. 48, No. 2, 2000 split injector was combined by direct coupling to a HewlettPackard 5972A mass spectrometer. The same type of column and the same temperature program as just described for GC were used. Helium at 1 mL/min was used as a carrier gas. The temperature of the ion source was 280 °C, and the electron energy was 70 eV. The electron impact (EI) mode was used. Infrared (IR) Spectroscopy. The IR spectra were obtained with a Perkin-Elmer 1760X spectrometer. CHCl3 was used as a solvent. Nuclear Magnetic Resonance (NMR) Spectroscopy. The NMR spectra were obtained with a JEOL GSX-270 (270.05 MHz, 1H; 67.80 MHz, 13C) spectrometer. Rearing of Larvae. The larvae of S. litura were reared in plastic cases (200 × 300 mm wide, 100 mm high, 100 larvae/ case) covered with a nylon mesh screen. The rearing conditions were as follows: 25 °C, 70% relative humidity, and constant light. A commercial diet (Insecta LF; Nihon Nosan Kogyo Co., Ltd.) was given to the larvae from the first instar. From the fourth instar, the diet was changed to an artificial diet composed of kidney beans (100 g), brewer’s dried yeast (40 g), ascorbic acid (4 g), agar (12 g), and water (600 mL; Yushima et al., 1991). Administration of β-Myrcene (1). The artificial diet without the agar was mixed with a blender. Two thousand milligrams of 1 was then added directly into the blender at 1 mg/g of diet. Agar was dissolved in water and boiled and then added into the blender. The diet was then mixed and cooled in a tray (220 × 310 mm wide, 30 mm high). The diet containing 1 was stored in a refrigerator until the time of administration. The last instar larvae (average weight ) 0.5 g) were moved into new cases (100 larvae/case), and the diet was fed to the larvae in limited amounts. Groups of 900 larvae were fed the diet containing 1 for 1 day, and then the artificial diet not containing 1 was fed to the larvae for an additional 1 day. Frass was collected daily (total of 2 days) and stored in a solution of CH2Cl2 (500 mL). For diet and frass separation, the fresh frass was extracted as soon as the last instar larvae excreted. Isolation and Identification of Metabolites from Frass. The frass was extracted three times with CH2Cl2 each time. The extract solution was evaporated under reduced pressure, and 2468 mg of extract was obtained. The extract was distributed between 5% NaHCO3 (aq) and CH2Cl2, the CH2Cl2 phase was evaporated, and the neutral fraction (1438 mg) was obtained. The neutral fraction was analyzed by GC-MS; metabolites 2 and 3 occurred in this fraction. The alkali phase was acidified with 1 N HCl and distributed between water and CH2Cl2. The CH2Cl2 phase was evaporated, and the acidic fraction (728 mg) was obtained. The neutral fraction was subjected to silica gel open-column chromatography (silica gel 60, 230-400 mesh, Merck) with a 9:1 n-hexane/EtOAc solvent system; 2 (417 mg) and 3 (96 mg) were isolated. Metabolites 2 and 3 were identified by a comparison of established MS, IR, and NMR data. Myrcene-3(10)-glycol (2): [R]D -0.33° (CHCl3, c 1); EIMS, m/z (rel intensity) 170 (M+, C10H18O2, trace) 152 (4), 139 (22), 121 (46), 69 (100); IR (νmax, cm-1) 3382, 3086, 1460, 1375, 1258, 1198, 1151, 925, 838; 1H NMR (CDCl3) δ 1.59 (3H, s, 8-CH3), 1.69 (3H, s, 9-CH3), 2.50 (2H, s, two OH), 3.47 (2H, s, H-10), 5.10 (1H, bt, H-6), 5.28 (1H, q, H-1), 5.36 (1H, q, H-1), 5.81 (1H, q, H-2); 13C NMR (CDCl3) δ 17.7 (C-9), 22.0 (C-5), 25.7 (C-8), 36.7 (C-4), 68.8 (C-10), 76.2 (C-3), 115.3 (C-1), 124.1 (C6), 132.3 (C-7), 140.6 (C-2). Myrcene-1,2-glycol (3): [R]D -5.4° (CHCl3, c 1); EIMS, m/z (rel intensity) 170(M+, C10H18O2, trace), 152 (2), 139 (22), 121 (46), 109 (26), 95 (15), 69 (100); IR (νmax, cm-1) 3375, 3398, 1018, 912, 839; 1H NMR (CDCl3) δ 1.63 (3H, s, 8-CH3), 1.70 (3H, s, 9-CH3), 3.63 (2H, m, OH), 4.16 (2H, s, OH), 5.12 (1H, m, CdCH); 13C NMR (CDCl3) δ 17.7 (C-9), 25.6 (C-8), 26.6 (C5), 32.8 (C-4), 66.0 (C-1), 75.3 (C-2), 110.6 (C-10), 124.1 (C-6), 131.8 (C-7), 148.2 (C-3). Incubation of Intestinal Bacteria with β-Myrcene (1). This experiment was carried out under sterile conditions. Petri dishes, pipets, and solutions were autoclaved. A GAM broth (Nissui Pharmaceutical Co., Ltd.) was adjusted to pH 9.0 and
Miyazawa and Murata placed in Petri dishes at 10 mL/Petri dish. The fresh frass (5 g) of the last instar larvae was suspended in physiological saline (100 mL), and the suspension (1 mL) was pipetted into the medium. The medium without frass was also prepared for a blank experiment. These media were incubated (18 °C, darkness, 2 days) under aerobic and anaerobic conditions. After growth of bacteria, 1 (10 mg/Petri dish) was added to the medium and the incubation was continued. The percentage of metabolites in the medium was determined 12, 24, and 48 h after addition of 1. The medium was acidified with 1 N HCl and distributed between Et2O and saturated solution of salt. The Et2O phase was evaporated, and the extract was obtained. For the quantitative analysis of metabolites, the GC analysis was used as an internal standard with 1. RESULTS AND DISCUSSION
Metabolites from Frass. Biotransformation by the larvae of S. litura was observed as follows: substrate was administered to the larvae through their diet; metabolites were then detected and isolated from the frass of larvae. In a previous paper, R-terpinene was mixed in the diet of larvae at a high concentration (10 mg/g of diet) to increase the production of potential metabolites (Miyazawa et al., 1996). Although alcohols were detected by GC analysis, intermediary metabolites (alcohols and aldehydes) were not isolated. This suggested that intermediary metabolites were only slightly excreted into the frass. In the present study, a concentration of 1 mg/g of diet was therefore chosen as optimum for administration. “Optimum” means the concentration results in complete consumption of substrate. The larvae that were fed the diet without substrate were used as control, and the extract of frass was analyzed by GC. No terpenoids were observed in the frass of controls. For the consumption of substrate in the diet observed, we varied the quantity of substrate in the diet by the internal standard method. The result was that consumption of β-myrcene (1) was 43%. In the biotransformation of 1, the two metabolites isolated from the frass were identified as 2 (48%) and 3 (9%). Percentage was calculated from the peak area in the GC spectra of the extract of frass: 100% was defined as total metabolites of 1. Substrate 1 and other compounds were detected in the frass by GC analysis. Metabolite 2 was produced by oxidation at the 3,10double bond of 1, and metabolite 3 was produced by oxidation at the 1,2-double bond of 1. Intestinal Bacteria. A previous paper described the participation of S. litura intestinal bacteria in the metabolism of R-terpinene (Miyazawa et al., 1996). The aerobically active intestinal bacteria transformed R-terpinene to p-mentha-1,3-dien-7-ol, and the anaerobically active intestinal bacteria transformed R-terpinene to p-cymene. In the present study, the in vitro metabolism of 1 by intestinal bacteria was also examined in a manner similar to that of the previous paper. However, 1 was not metabolized at all (no reaction). These results suggested that the intestinal bacteria did not participate in the metabolism of 1. The difference of reaction between 1 and R-terpinene was suggested to be due to the difference of substrate. Metabolic Pathways. In the present study of biotransformation of 1, the larvae transformed 1 to 2 and 3 (Scheme 1). The double bonds of 1 were preferentially oxidized as in the biotransformation of (+)- and (-)-limonene. These results indicate double bonds are preferentially oxidized. Compound 1 is the most important and widespread terpene known; however, there are a few studies on the
Biotransformation of β-Myrcene by Cutworm Larvae Scheme 1. Possible Metabolic β-Myrcene (1) by S. litura
J. Agric. Food Chem., Vol. 48, No. 2, 2000 125 Pathways
of
biotransformation of 1 by other organisms. Compound 1 appears to produce different metabolites with different species of organisms. However, species of microorganisms or mammals have similar metabolic pathways (oxidative positions). The oxidations at the 3(10)- and 1,2-double bonds are the main metabolic pathways in the biotransformation of 1 by rabitt (Ishida et al., 1981); the oxidations at each of these three double bonds are the main metabolic pathways in the biotransformation of 1 by Aspergillus niger (Yamazaki et al., 1988). In the biotransformation of 1 in rabbit, compounds 2 and 3 are produced; compound 2 is the main product. On the other hand, in the biotransformation of 1 by A. niger, compounds 2 and 3 and myrcene-6,7-glycol are produced; myrcene-6,7-glycol is the main product. In the present study, the main product converted by the larvae of S. litura is the same as in rabbit; therefore, the larvae of S. litura employ a metabolic pathway similar to that used by the rabbit.
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Received for review April 23, 1999. Accepted November 10, 1999. JF990398M
